The present invention relates generally to signal transduction pathways. More particularly, the present invention relates to chemokine receptors, nucleic acids encoding chemokine receptors, chemokine receptor ligands, modulators of chemokine receptor activity, antibodies recognizing chemokines and chemokine receptors, methods for identifying chemokine receptor ligands and modulators, methods for producing chemokine receptors, and methods for producing antibodies recognizing chemokine receptors.
Recent advances in molecular biology have led to an appreciation of the central role of signal transduction pathways in biological processes. These pathways comprise a central means by which individual cells in a multicellular organism communicate, thereby coordinating biological processes. See Springer, Cell 76:301-314 (1994), Table I for a model. One branch of signal transduction pathways, defined by the intracellular participation of guanine nucleotide binding proteins (G-proteins), affects a broad range of biological processes.
Lewin, GENES V319-348 (1994) generally discusses G-protein signal transduction pathways which involve, at a minimum, the following components: an extracellular signal (e.g., neurotransmitters, peptide hormones, organic molecules, light, or odorants), a signal-recognizing receptor (G-protein-coupled receptor, reviewed in Probst et al., DNA and Cell Biology 11:1-20 [1992] and also known as GPR or GPCR), and an intracellular, heterotrimeric GTP-binding protein, or G protein. In particular, these pathways have attracted interest because of their role in regulating white blood cell or leukocyte trafficking.
Leukocytes comprise a group of mobile blood cell types including granulocytes (i.e., neutrophils, basophils, and eosinophils), lymphocytes, and monocytes. When mobilized and activated, these cells are primarily involved in the body""s defense against foreign matter. This task is complicated by the diversity of normal and pathological processes in which leukocytes participate. For example, leukocytes function in the normal inflammatory response to infection. Leukocytes are also involved in a variety of pathological inflammations. For a summary, see Schall et al., Curr. Opin. Immunol. 6:865-873 (1994). Moreover, each of these processes can involve unique contributions, in degree, kind, and duration, from each of the leukocyte cell types.
In studying these immune reactions, researchers initially concentrated on the signals acting upon leukocytes, reasoning that a signal would be required to elicit any form of response. Murphy, Ann. Rev. Immunol. 12:593-633 (1994) has reviewed members of an important group of leukocyte signals, the peptide signals. One type of peptide signal comprises the chemokines (chemoattractant cytokines), termed intercrines in Oppenheim et al., Ann. Rev. Immunol. 9:617-648 (1991). In addition to Oppenheim et al., Baggiolini et al., Advances in Immunol. 55:97-179 (1994), documents the growing number of chemokines that have been identified and subjected to genetic and biochemical analyses.
Comparisons of the amino acid sequences of the known chemokines have led to a classification scheme which divides chemokines into two groups: the xcex1 group characterized by a single amino acid separating the first two cysteines (CXC; N-terminus as referent), and the xcex2 group, where these cysteines are adjacent (CC). See Baggiolini et al., supra. Correlations have been found between the chemokines and the particular leukocyte cell types responding to those signals. Schall et al., supra, has reported that the CXC chemokines generally affect neutrophils; the CC chemokines tend to affect monocytes, lymphocytes, basophils and eosmophils. For example, Baggiolini et al., supra, recited that RANTES, a CC chemokine, functions as a chemoattractant for monocytes, lymphocytes (i.e., memory T cells), basophils, and eosinophils, but not for neutrophils, while inducing the release of histamine from basophils.
Chemokines were recently shown by Cocchi, et. al., Science, 270: 1811-1815 (1995) to be suppressors of HIV proliferation. Cocchi, et al. demonstrated that RANTES, MIP-1xcex1, and MIP-1xcex2 suppressed HIV-1, HIV-2 and SIV infection of a CD4+ cell line designated PM1 and of primary human peripheral blood mononuclear cells.
Recently, however, attention has turned to the cellular receptors that bind the chemokines, because the extracellular chemokines seem to contact cells indiscriminately, and therefore lack the specificity needed to regulate the individual leukocyte cell types.
Murphy, supra, reported that the GPCR superfamily of receptors includes the chemokine receptor family. The typical chemokine receptor structure includes an extracellular chemokine-binding domain located near the N-terminus, followed by seven spaced regions of predominantly hydrophobic amino acids capable of forming membrane-spanning xcex1-helices. Between each of the xcex1-helical domains are hydrophilic domains localized, alternately, in the intra- or extra-cellular spaces. These features impart a serpentine conformation to the membrane-embedded chemokine receptor. The third intracellular loop typically interacts with G-proteins. In addition, Murphy, supra, noted that the intracellular carboxyl terminus is also capable of interacting with G-proteins.
The first chemokine receptors to be analyzed by molecular cloning techniques were the two neutrophil receptors for human IL8, a CXC chemokine. Holmes et al., Science 253:178-1280 (1991) and Murphy et al., Science 253:1280-1283 (1991), reported the cloning of these two receptors for IL8. Lee et al., J. Biol. Chem. 267:16283-16287 (1992), analyzed the cDNAs encoding these receptors and found 77% amino acid identity between the encoded receptors, with each receptor exhibiting features of the G protein coupled receptor family. One of these receptors is specific for IL-8, while the other binds and signals in response to IL-8, gro/MGSA, and NAP-2. Genetic manipulation of the genes encoding IL-8 receptors has contributed to our understanding of the biological roles occupied by these receptors. For example, Cacalano et al., Science 265:682-684 (1994) reported that deletion of the IL-8 receptor homolog in the mouse resulted in a pleiotropic phenotype involving lymphadenopathy and splenomegaly. In addition, a study of missense mutations described in Leong et al., J. Biol. Chem. 269:19343-19348 (1994) revealed amino acids in the IL-8 receptor that were critical for IL-8 binding. Domain swapping experiments discussed in Murphy, supra, implicated the amino terminal extracellular domain as a determinant of binding specificity.
Several receptors for CC chemokines have also been identified and cloned. CCCKR1 binds both MIP-1xcex1 and RANTES and causes intracellular calcium ion flux in response to both ligands. Charo et al., Proc Natl. Acad. Sci. (USA) 91:2752-2756 (1994) reported that another CC chemokine receptor, MCP-R1 (CCCKR2), is encoded by a single gene that produces two splice variants which differ in their carboxyl terminal domains. This receptor binds and responds to MCP-3 in addition to MCP-1.
A promiscuous receptor that binds both CXC and CC chemokines has also been identified. This receptor was originally identified on red blood cells and Horuk et al., Science 261:1182-1184 (1993) reports that it binds IL-8, NAP-2, GROxcex1, RANTES, and MCP-1. The erythrocyte chemokine receptor shares about 25% identity with other chemokine receptors and may help to regulate circulating levels of chemokines or aid in the presentation of chemokines to their targets. In addition to binding chemokines, the erythrocyte chemokine receptor has also been shown to be the receptor for plasmodium vivax, a major cause of malaria (id.) Another G-protein coupled receptor which is closely related to chemokine receptors, the platelet activating factor receptor, has also been shown to be the receptor for a human pathogen, the bacterium Streptococcus pneumoniae (Cundell et al., Nature 377:435-438 (1995)).
In addition to the mammalian chemokine receptors, two viral chemokine receptor homologs have been identified. Ahuja et al., J. Biol. Chem. 268:20691-20694 (1993) describes a gene product from Herpesvirus saimiri that shares about 30% identity with the IL-8 receptors and binds CXC chemokines. Neote et al., Cell, 72:415-425 (1993) reports that human cytomegalovirus contains a gene encoding a receptor sharing about 30% identity with the CC chemokine receptors which binds MIP-1xcex1, MIP-1xcex2, MCP-1, and RANTES. These viral receptors may affect the normal role of chemokines and provide a selective pathological advantage for the virus.
Because of the broad diversity of chemokines and their activities, there are numerous receptors for the chemokines. The receptors which have been characterized represent only a fraction of the total complement of chemokine receptors. There thus remains a need in the art for the identification of additional chemokine receptors. The availability of these novel receptors will provide tools for the development of therapeutic modulators of chemokine or chemokine receptor function. It is contemplated by the present invention that such modulators are useful as therapeutics for the treatment of atherosclerosis, rheumatoid arthritis, tumor growth suppression, asthma, viral infections, and other inflammatory conditions. Alternatively, fragments or variants of the chemokine receptors, or antibodies recognizing those receptors, are contemplated as therapeutics.
The present invention provides purified and isolated nucleic acids encoding chemokine receptors involved in leukocyte trafficking. Polynucleotides of the invention (both sense and anti-sense strands thereof) include genomic DNAs, cDNAs, and RNAs, as well as completely or partially synthetic nucleic acids. Preferred polynucleotides of the invention include the DNA encoding the chemokine receptor 88-2B that is set out in SEQ ID NO:3, the DNA encoding the chemokine receptor 88C that is set out in SEQ ID NO:1, and DNAs which hybridize to those DNAs under standard stringent hybridization conditions, or which would hybridize but for the redundancy of the genetic code Exemplary stringent hybridization conditions are as follows: hybridization at 42xc2x0 C. in 50% formamide, 5xc3x97SSC, 20 mM sodium phosphate, pH 6.8 and washing in 0.2xc3x97SSC at 55xc2x0 C. It is understood by those of skill in the art that variation in these conditions occurs based on the length and GC nucleotide content of the sequences to be hybridized. Formulas standard in the art are appropriate for determining exact hybridization conditions. See Sambrook et al., xc2xa7xc2xa7 9.47-9.51 in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Also contemplated by the invention are polynucleotides encoding domains of 88-2B or 88C, for example, polynucleotides encoding one or more extracellular domains of either protein or other biologically active fragments thereof. 88-2B extracellular domains correspond to SEQ ID NO:3 and SEQ ID NO:4 at amino acid residues 1-36, 93-107, 171-196, and 263-284. The extracellular domains of 88-2B are encoded by polynucleotide sequences corresponding to SEQ ID NO:3 at nucleotides 362-469, 638-682, 872-949, and 1148-1213. Extracellular domains of 88C correspond to SEQ ID NO:1 and SEQ ID NO:2 at amino acid residues 1-32, 89-112, 166-191, and 259-280. The 88C extracellular domains are encoded by polynucleotide sequences that correspond to SEQ ID NO:1 at nucleotides 55-150, 319-390, 550-627, and 829-894. The invention also comprehends polynucleotides encoding intracellular domains of these chemokine receptors. The intracellular domains of 88-2B include amino acids 60-71, 131-151, 219-240, and 306-355 of SEQ ID NO:3 and SEQ ID NO:4. Those domains are encoded by polynucleotide sequences corresponding to SEQ ID NO:3 at nucleotides 539-574, 752-814, 1016-1081, and 1277-1426, respectively. The 88C intracellular domains include amino acid residues 56-67, 125-145, 213-235, and 301-352 of SEQ ID NO:1 and SEQ ID NO:2. The intracellular, domains of 88C are encoded by polynucleotide sequences corresponding to SEQ ID NO:1 at nucleotides 220-255, 427-489, 691-759, and 955-1110. Peptides corresponding to one or more of the extracellular or intracellular domains, or antibodies raised against those peptides, are contemplated as modulators of receptor activities, especially ligand and G protein binding activities of the receptors.
The nucleotide sequences of the invention may also be used to design oligonucleotides for use as labeled probes to isolate genomic DNAs encoding 88-2B or 88C under stringent hybridization conditions (i.e., by Southern analyses and Polymerase Chain Reaction methodologies). Moreover, these oligonucleotide probes can be used to detect particular alleles of the genes encoding 88-2B or 88C, facilitating both diagnosis and gene therapy treatments of disease states associated with particular alleles. In addition, these oligonucleotides can be used to alter chemokine receptor genetics to facilitate identification of chemokine receptor modulators. Also, the nucleotide sequences can be used to design antisense genetic elements of use in exploring or altering the genetics and expression of 88-2B or 88C. The invention also comprehends biological replicas (i.e., copies of isolated DNAs made in vivo or in vitro) and RNA transcripts of DNAs of the invention. Autonomously replicating recombinant constructions such as plasmid, viral, and chromosomal (e.g., YAC) nucleic acid vectors effectively incorporating 88-2B or 88C polynucleotides, and, particularly, vectors wherein DNA effectively encoding 88-2B or 88C is operatively linked to one or more endogenous or heterologous expression control sequences are also provided.
The 88-2B and 88C receptors may be produced naturally, recombinantly or synthetically. Host cells (prokaryotic or eukaryotic) transformed or transfected with polynucleotides of the invention by standard methods may be used to express the 88-2B and 88C chemokine receptors. Beyond the intact 88-2B or 88C gene products, biologically active fragments of 88-2B or 88C, analogs of 88-2B or 88C, and synthetic peptides derived from the amino acid sequences of 88-2B, set out in SEQ ID NO:4, or 88C, set out in SEQ ID NO:2, are contemplated by the invention. Moreover, the 88-2B or 88C gene product, or a biologically active fragment of either gene product, when produced in a eukaryotic cell, may be post-translationally modified (e.g., via disulfide bond formation, glycosylation, phosphorylation, myristoylation, palmitoylation, acetylation, etc.) The invention further contemplates the 88-2B and 88C gene products, or biologically active fragments thereof, in monomeric, homomultimeric, or heteromultimeric conformations.
In particular, one aspect of the invention involves antibody products capable of specifically binding to the 88-2B or 88C chemokine receptors. The antibody products are generated by methods standard in the art using recombinant 88-2B or 88C receptors, synthetic peptides or peptide fragments of 88-2B or 88C receptors, host cells expressing 88-2B or 88C on their surfaces, or 88-2B or 88C receptors purified from natural sources as immunogens. The antibody products may include monoclonal antibodies or polyclonal antibodies of any source or sub-type. Moreover, monomeric, homomultimeric, and heteromultimeric antibodies, and fragments thereof, are contemplated by the invention. Further, the invention comprehends CDR-grafted antibodies, xe2x80x9chumanizedxe2x80x9d antibodies, and other modified antibody products retaining the ability to specifically bind a chemokine receptor.
The invention also contemplates the use of antibody products for detection of the 88-2B or 88C gene products, their analogs, or biologically active fragments thereof. For example, antibody products may be used in diagnostic procedures designed to reveal correlations between the expression of 88-2B, or 88C, and various normal or pathological states. In addition, antibody products can be used to diagnose tissue-specific variations in expression of 88-2B or 88C, their analogs, or biologically active fragments thereof. Antibody products specific for the 88-2B and 88C chemokine receptors may also act as modulators of receptor activities. In another aspect, antibodies to 88-2B or 88C receptors are useful for therapeutic purposes.
Assays for ligands capable of interacting with the chemokine receptors of the invention are also provided. These assays may involve direct detection of chemokine receptor activity, for example, by monitoring the binding of a labeled ligand to the receptor. In addition, these assays may be used to indirectly assess ligand interaction with the chemokine receptor. As used herein the term xe2x80x9cligandxe2x80x9d comprises molecules which are agonists and antagonists of 88-2B or 88C, and other molecules which bind to the receptors.
Direct detection of ligand binding to a chemokine receptor may be achieved using the following assay. Test compounds (i.e., putative ligands) are detectably labeled (e.g., radioiodinated). The detectably labeled test compounds are then contacted with membrane preparations containing a chemokine receptor of the invention. Preferably, the membranes are prepared from host cells expressing chemokine receptors of the invention from recombinant vectors. Following an incubation period to facilitate contact between the membrane-embedded chemokine receptors and the detectably labeled test compounds, the membrane material is collected on filters using vacuum filtration. The detectable label associated with the filters is then quantitated. For example, radiolabels are quantitated using liquid scintillation spectrophotometry. Using this technique, ligands binding to chemokine receptors are identified. To confirm the identification of a ligand, a detectably labeled test compound is exposed to a membrane preparation displaying a chemokine receptor in the presence of increasing quantities of the test compound in an unlabeled state. A progressive reduction in the level of filter-associated label as one adds increasing quantities of unlabeled test compound confirms the identification of that ligand.
Agonists are ligands which bind to the receptor and elicit intracellular signal transduction and antagonists are ligands which bind to the receptor but do not elicit intracellular signal transduction. The determination of whether a particular ligand is an agonist or an antagonist can be determined, for example, by assaying G protein-coupled signal transduction pathways. Activation of these pathways can be determined by measuring intracellular ca++ flux, phospholipase C activity or adenylyl cyclase activity, in addition to other assays (see examples 5 and 6).
As discussed in detail in the Examples herein, chemokines that bind to the 88C receptor include RANTES, MIP-1xcex1, and MIP-1xcex2, and chemokines that bind to the 88-2B receptor include RANTES.
In another aspect, modulators of the interaction between the 88C and 88-2B receptors and their ligands are specifically contemplated by the invention. Modulators of chemokine receptor function may be identified using assays similar to those used for identifying ligands. The membrane preparation displaying a chemokine receptor is exposed to a constant and known quantity of a detectably labeled functional ligand. In addition, the membrane-bound chemokine receptor is also exposed to an increasing quantity of a test compound suspected of modulating the activity of that chemokine receptor. If the levels of filter-associated label correlate with the quantity of test compound, that compound is a modulator of the activity of the chemokine receptor. If the level of filter-associated label increases with increasing quantities of the test compound, an activator has been identified. In contrast, if the level of filter-associated label varies inversely with the quantity of test compound, an inhibitor of chemokine receptor activity has been identified. Testing for modulators of receptor binding in this way allows for the rapid screening of many putative modulators, as pools containing many potential modulators can be tested simultaneously in the same reaction.
The indirect assays for receptor binding involve measurements of the concentration or level of activity of any of the components found in the relevant signal transduction pathway. Chemokine receptor activation often is associated with an intracellular Ca++ flux. Cells expressing chemokine receptors may be loaded with a calcium-sensitive dye. Upon activation of the expressed receptor, a Ca++ flux would be rendered spectrophotometrically detectable by the dye. Alternatively, the Ca++ flux could be detected microscopically. Parallel assays, using either technique, may be performed in the presence and absence of putative ligands. For example, using the microscopic assay for Ca++ flux, RANTES, a CC chemokine, was identified as a ligand of the 88-2B chemokine receptor. Those skilled in the art will recognize that these assays are also useful for identifying and monitoring the purification of modulators of receptor activity. Receptor activators and inhibitors will activate or inhibit, respectively, the interaction of the receptors with their ligands in these assays.
Alternatively, the association of chemokine receptors with G proteins affords the opportunity of assessing receptor activity by monitoring G protein activities. A characteristic activity of G proteins, GTP hydrolysis, may be monitored using, for example, 32P-labeled GTP.
G proteins also affect a variety of other molecules through their participation in signal transduction pathways. For example, G protein effector molecules include adenylyl cyclase, phospholipase C, ion channels, and phosphodiesterases. Assays focused on any of these effectors may be used to monitor chemokine receptor activity induced by ligand binding in a host cell that is both expressing the chemokine receptor of interest and contacted with an appropriate ligand. For example, one method by which the activity of chemokine receptors may be detected involves measuring phospholipase C activity. In this assay, the production of radiolabeled inositol phosphates by host cells expressing a chemokine receptor in the presence of an agonist is detected. The detection of phospholipase activity may require cotransfection with DNA encoding an exogenous G protein. When cotransfection is required, this assay can be performed by cotransfection of chimeric G protein DNA, for example, Gqi5 (Conklin, et al., Nature 363:274-276 (1993), with 88-2B or 88C DNA and detecting phosphoinositol production when the cotransfected cell is exposed to an agonist of the 88-2B or 88C receptor. Those skilled in the art will recognize that assays focused on G-protein effector molecules are also useful for identifying and monitoring the purification of modulators of receptor activity. Receptor activators and inhibitors will activate or inhibit, respectively, the interaction of the receptors with their ligands in these assays.
Chemokines have been linked to many inflammatory diseases, such as psoriasis, arthritis, pulmonary fibrosis and atherosclerosis. See Baggiolini et al., supra. Inhibitors of chemokine action may be useful in treating these conditions. In one example, Broaddus et al., J. of Immunol. 152:2960-2967 (1994), describes an antibody to IL-8 which can inhibit neutrophil recruitment in endotoxin-induced pleurisy, a model of acute inflammation in rabbit lung. It is also contemplated that ligand or modulator binding to, or the activation of, the 88C receptor may be useful in treatment of HIV infection and HIV related disease states. Modulators of chemokine binding to specific receptors contemplated by the invention may include antibodies directed toward a chemokine or a receptor, biological or chemical small molecules, or synthetic peptides corresponding to fragments of the chemokine or receptor.
Administration of compositions containing 88-2B or 88C modulators to mammalian subjects, for the purpose of monitoring or remediating normal or pathological immune reactions And viral infections including infection by retroviruses such as HIV-1, HIV-2 and SIV is contemplated by the invention. In particular, the invention comprehends the mitigation of inflammatory responses, abnormal hematopoietic processes, and viral infections by delivery of a pharmaceutically acceptable quantity of 88-2B or 88C chemokine receptor modulators. The invention further comprehends delivery of these active substances in pharmaceutically acceptable compositions comprising carriers, diluents, or medicaments. The invention also contemplates a variety of administration routes. For example, the active substances may be administered by the following routes: intravenous, subcutaneous, intraperitoneal, intramuscular, oral, anal (i.e., via suppository formulations), or pulmonary (i.e., via inhalers, atomizers, nebulizers, etc.)
In another aspect, the DNA sequence information provided by the present invention makes possible the development, by homologous recombination or xe2x80x9cknockoutxe2x80x9d strategies [see, e.g. Kapecchi, Science, 244:1288-1292 (1989)], of rodents that fail to express a functional 88C or 88-2B -chemokine receptor or that express a variant of the receptor. Alternatively, transgenic mice which express a cloned 88-2B or 88C receptor can be prepared by well known laboratory techniques (Manipulating the Mouse Embryo: A Laboratory Manual, Brigid Hohan, Frank Costantini and Elizabeth Lacy, eds. (1986) Cold Spring Harbor Laboratory ISBN 0-87969-175-I). Such rodents are useful as models for studying the activities of 88C or 88-2B receptors in vivo.
Other aspects and advantages of the present invention will become apparent to one skilled in the art upon consideration of the following examples.